Microbial targets for protective humoral immunity are typically surface-localized proteins and contain common sequence motifs related to their secretion or surface binding. Exploiting the whole genome sequence of the human bacterial pathogen Streptococcus pneumoniae, we identified 130 open reading frames encoding proteins with secretion motifs or similarity to predicted virulence factors. Mice were immunized with 108 of these proteins, and 6 conferred protection against disseminated S. pneumoniae infection. Flow cytometry confirmed the surface localization of several of these targets. Each of the six protective antigens showed broad strain distribution and immunogenicity during human infection. Our results validate the use of a genomic approach for the identification of novel microbial targets that elicit a protective immune response. These new antigens may play a role in the development of improved vaccines against S. pneumoniae.Streptococcus pneumoniae (the pneumococcus) is the leading cause of bacterial sepsis, pneumonia, meningitis, and otitis media in young children in the United States. Annually, 7,000,000 middle-ear infections are ascribed to this organism (4). The vaccines in current use are formulations of capsular carbohydrate from the 23 serotypes responsible for 85 to 90% of infections in the United States, but these vaccines are poorly efficacious in infants and the elderly, the populations that are most at risk (1). A heptavalent-capsular-carbohydrate vaccine conjugated to the protein carrier CRM197 has been shown to be well tolerated and efficacious against invasive disease caused by the seven vaccine serotype strains (3) and has recently been approved for use in young children. However, this type of vaccine has several potential limitations, including serotype replacement by strains that are not represented (14).The advent of whole-genome sequencing of microbes, including microbial pathogens, has revolutionized the methods by which these organisms are studied and has heightened expectations regarding the ability to predict potential targets for antimicrobial agents and vaccines (2,12,20). We combined sequence scanning for prediction of surface-localized proteins with an animal model which allowed us to directly screen proteins for vaccine efficacy to identify novel vaccine candidates from the genome sequence of S. pneumoniae. Here we describe the use of a clinically relevant animal model for the evaluation of the vaccine efficacy of proteins identified from the genome sequence of pneumococcus. This approach was validated by the discovery of five previously unidentified genes whose products induced immune responses that protected mice from pneumococcal infection. Similar sequence scanning methods were recently used to identify potential vaccine candidates from the genomic sequence of the gram-negative pathogen Neisseria meningitidis (21) predicted by in vitro correlates of vaccine effectiveness. Here we expand upon the use of genomics to directly demonstrate vaccine efficacy in an animal model for...
Pathogenic bacteria rely on adhesins to bind to host tissues. Therefore, the maintenance of the functional properties of these extracellular macromolecules is essential for the pathogenicity of these microorganisms. We report that peptide methionine sulfoxide reductase (MsrA), a repair enzyme, contributes to the maintenance of adhesins in Streptococcus pneumoniae, Neisseria gonorrhoeae, and Escherichia coli. A screen of a library of pneumococcal mutants for loss of adherence uncovered a MsrA mutant with 75% reduced binding to GalNAcI81-4Gal containing eukaryotic cell receptors that are present on type II lung cells and vascular endothelial cells. Subsequently, it was shown that an E. coli msrA mutant displayed decreased type I fimbriae-mediated, mannosedependent, agglutination of erythrocytes. Previous work [Taha,
Blocking the primary stages of infection, namely bacterial attachment to host cell receptors and colonization of the mucosal surface, may be the most effective strategy to prevent bacterial infections. Bacterial attachment usually involves an interaction between a bacterial surface protein called an adhesin and the host cell receptor. Recent preclinical vaccine studies with the FimH adhesin (derived from uropathogenic Escherichia coli) have confirmed that antibodies elicited against an adhesin can impede colonization, block infection, and prevent disease. The studies indicate that prophylactic vaccination with adhesins can block bacterial infections. With recent advances in the identification, characterization, and isolation of other adhesins, similar approaches are being explored to prevent infections, from otitis media and dental caries to pneumonia and sepsis.
Nitric oxide is a short-lived cytotoxic mediator that has been implicated in the pathogenesis of endotoxin-induced tissue injury and septic shock. In the present studies we determined whether this mediator is produced in the lung during acute endotoxemia. We found that intravenous injection of rats with bacterially derived lipopolysaccharide (LPS), a condition that induces acute endotoxemia, caused a time-dependent increase in inducible nitric oxide synthase (iNOS) mRNA expression in the lung, which reached a maximum after 24 h. This was correlated with nitric oxide production in the lung as measured by electron paramagnetic spin trapping, which was detectable within 6 h. Alveolar macrophages (AMs) and interstitial macrophages (IMs) isolated from rats 6-12 h after induction of acute endotoxemia were also found to exhibit increased nitric oxide production in response to in vitro stimulation with interferon-gamma (IFN-gamma) and LPS measured by nitrite accumulation in the culture medium. The effects of acute endotoxemia on nitric oxide production by these cells were, however, transient and returned to control levels by 24 h in AMs and 36 h in IMs. Interestingly, although nitrite accumulation in the culture medium of IMs isolated 48 h after induction of acute endotoxemia and stimulated with low concentrations of IFN-gamma and LPS was reduced, when compared with cells from control animals, these cells, as well as AMs, continued to express high levels of iNOS protein and mRNA. This was correlated with increased peroxynitrite production by the cells. Peroxynitrite has been shown to act as a nitrating agent and can generate nitrotyrosine residues in proteins. Using a specific antibody and immunohistochemistry, we found evidence of nitrotyrosine residues in sections of lungs 48 h after treatment of rats with endotoxin. These data suggest that nitric oxide produced by IMs and AMs can react with superoxide anion to form peroxynitrite. Taken together, the present studies demonstrate that AMs and IMs are activated following acute endotoxemia to produce reactive nitrogen intermediates and that both cell types contribute to inflammatory responses in the lung.
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